In biotechnology and genetic research, it is often desirable to use marker genes or proteins to test and experiment with genetic processes and gene expression methodologies. For example, if one seeks to test a gene transfection process, the first gene that is usually employed is a test gene the expression of which can easily be seen in the host. Thus, genes that encode colorimetric and fluorescent proteins have become popular tools for genetic and gene expression research.
One of the popular fluorescent markers that is used in this fashion is the green fluorescent protein (GFP). The GFP protein, originally isolated from the jellyfish Aequorea, absorbs light in the visible spectrum and fluoresces in a visible green shade. It has been found that the GFP protein can be expressed in many different hosts and organisms while still retaining the characteristic fluorescent activity. One common use of GFP is to place the GFP gene in tandem with some other gene so that the fluorescence of treated cells will indicate the presence and expression of transferred DNA.
The GFP protein is a robust and stable molecule. It forms a barrel-shaped tertiary structure with a series of beta sheets forming a stable cylinder with the three amino acid chromophore region formed in its interior. The exterior surface of the barrel shape of the proteins is natively anionic, but exhibits a mixture of charged residues to its environment.
Since GFP has been in use as a research tool, many variants of GFP have been developed for a variety of purposes. However, most of the work on the GFP molecule has been on the fluorescent region of the protein to change the spectral characteristics of the fluorescence or to enhance the amplitude of the fluorescence. The characteristic of cell permeability is not usually considered with regard to fluorescent proteins like GFP. Indeed, breaching the plasma membrane barrier is a limiting factor in the development of proteins and other biomolecules as therapeutics and diagnostic tools. (See Marafino, B. J., Jr. & Pugsley, M. K. Cardiovasc. Toxicol. 3, 5-12 (2003)). Accordingly, there is much interest in developing new means to deliver proteins and other macromolecules into cells.